Among the complex network of cytokines that influence odontoblast function during development and repair, TGF-beta1 is unique in its dual abilities to function as a potent immunosuppressant and as an inducer of extracellular matrix (ECM) production. The molecular mechanisms of TGF-beta1 action during dentin formation are poorly understood. To better understand the precise physiologic functions of TGF-beta1 in dentinogenesis we have studied mice with a loss-of-function mutation at the TGF-beta1 locus. TGF-beta 1(-/-) mice die at weaning from widespread inflammation due to the loss of a critical regulator of immune function. Our studies of adult denition in TGF-beta1 (-/-) mice kept alive on dexamethasone show extensive pulp and periapical pathology and a marked attrition of occlusal surfaces. Teeth of TGF-beta1(-/-) mice backcrossed onto immunodeficient backgrounds also showed significant changes in dentin likely caused by defects in mineralization. The impairment in dentinogenesis is thus linked directly to the loss of TGF-beta1 rather than to the secondary effects of inflammation. In the proposed studies, TGF-beta-1(-/-) athymic nude mice will be used to rigorously test the hypothesis that TGF-beta1 is important for odontoblast function during primary and reparative dentin formation. In Aim 1, the structure and integrity of primary dentin will be assessed in TGF-beta1(-/-) nude mice by tetracycline labeling, Fourier transform infrared spectroscopy, ultrastructural and biomechanical approaches. Aim 2 will use cellular and molecular approaches to assess whether the absence of TGF-beta1 affects the expression of alkaline phosphatase and key dentin ECM genes, using in situ hybridization, RT-PCR and immunolocalization analyses. The biosynthetic abilities of odontoblasts will be tested by labeling organ cultures with 3H-proline and 45Ca. In Aim 3, the genetic pathways involving TGF-beta1 will be explored. The expression and functional relationship of genes like Bmp2 and Msx2 that may potentially interact with TGF-beta1 will be assessed in TGF-beta1(-/-) nude and Msx2(-/-) mice whose teeth also appear poorly mineralized. Studies in Aim 4 will assess whether TGF-beta1(-/-) odontoblasts can form reparative dentin in vivo. The effects of exogeneous TGF-beta1 placed at sites of pulp exposure will be compared in TGF-beta1(-/-) vs. TGF-beta1(+/-) nude littermates. The overall goal of these studies is to provide definitive information on TGF-beta1's role in dentin formation. Such insights will contribute to the development of bioactive dental materials to treat and prevent injuries of the dentin-pulp complex.